The present invention relates to the acylation of proteins. More particularly, the invention relates to a one-step process for selectively acylating the xcex5-amino group of proinsulin, insulin or an insulin analog in the presence of a free xcex1-amino group.
The acylation of amino groups is one of the most common means employed for chemically modifying proteins. General methods of acylation are set forth in Methods of Enzymology, 25:494-499 (1972) and include the use of activated esters, acid halides, or acid anhydrides. The use of activated esters, in particular N-hydroxysuccinimide esters of fatty acids is a particularly advantageous means of acylating a free amino acid with a fatty acid. Lapidot et al., J. of Lipid Res. 8:142-145 (1967). Lapidot et al. describe the preparation of N-hydroxysuccinimide esters and their use in the preparation of N-lauroyl-glycine, N-lauroyl-L-serine, and N-lauroyl-L-glutamic acid.
Early studies of selectively acylating the amino groups of insulin are described in Lindsay et al., in Biochem. J. 121:737-745 (1971). Lindsay et al., describe the reactivity of insulin with N-succinimidyl acetate at low reagent concentration and near neutral pH as producing two mono-substituted products. PheB1-acetyl-insulin and GlyA1-acetyl insulin. At pH 8.5, the amount of PheB1-acetyl insulin produced is lowered and LysB29-acetyl-insulin is also produced. Thus, Lindsay et al., conclude at pH 6.9 the order of reactivity is Glycine(A1)≈Phenylalanine(B1) greater than  greater than Lysine (B29) and at pH 8.5 Glycine(A1) greater than Phenylalanine=Lysine (B29). Id.
Lindsay et al., U.S. Pat. No. 3,869,437, disclose the acylation of the B1 amino acid with an acyl group containing up to seven carbons and optionally blocking the A1- and/or B29-amino group with an acyl group with up to four carbons. N-hydroxysuccinimide esters are described as particularly advantageous acylating agents. In order to produce the maximum yield of insulin acylated at the B1-amino group, the proportion of acylating agent is relatively low (one to not more than two molar equivalents of acylating agent). In addition, the maximum yield of mono-substituted B1 product is produced at a pH at or near about pH 7. At pH 8.5 to 9.0, the yield of the desired B1 acylated product falls off considerably in favor of additional substitution at positions A1 and B29.
D. G. Smyth, in U.S. Pat. No. 3,868,356 and Smyth et al., in U.S. Pat. No. 3,868,357 disclose N-acylated, O-substituted insulin derivatives in which at least one of the A1, B1 or B29 amino acid amino groups is converted into a blocked amino group. The acylation is carried out with a relatively small excess of acylating agent, e.g., from 2 to 3 moles per amino group at a neutral or mildly alkaline pH, e.g., 7-8. The reaction proceeds in very high yield with the formation of the di-substituted derivative resulting from the reaction of the A1- and B1-amino groups. In the presence of excess acylating agent, e.g., up to 10 molar, the reaction proceeds additionally at the B29-amino group to form the tri-substituted derivative.
To selectively acylate insulin, Muranishi and Kiso, in Japanese Patent Application 1-254,699, disclose a five-step synthesis for preparing fatty acid insulin derivatives. Step one, the activated fatty acid ester is prepared; Step two, the amino groups of insulin are protected with p-methoxy benzoxy carbonylazide (pMZ); Step three, the insulin-pMZ is reacted with the fatty acid ester; Step four, the acylated insulin is deprotected; and Step five, the acylated insulin is isolated and purified. Most notably, selective acylation of one amino group is only achieved by using the pMZ blocking group to protect the other amino groups. Using this methodology, Muranishi and Kiso prepare the following compounds: LysB29-palmitoyl insulin (the xcex5-amino group is acylated), PheB1-palmitoyl insulin (the N terminal xcex1-amino group of the B chain is acylated), and PheB1, LysB29-dipalmitoyl insulin (both the xcex5-amino and the N-terminal xcex1-amino group are acylated).
Similarly, Hashimoto et al., in Pharmaceutical Research 6:171-176 (1989), teach a four step synthesis for preparing N-palmitolyl insulin. The synthesis includes protecting and deprotecting the N-terminal. A1-glycine and the xcex5-amino group of B29-lysine, with pMZ. Under the conditions described in the reference, two major acylated products are prepared, B1-mono-palmitoyl insulin and B1, B29-dipalmitoyl insulin.
Therefore, prior to the present invention, the selective acylation of the B29-Nxcex5-amino group of insulin was carried out by protecting and subsequently deprotecting the xcex1-amino groups. The present invention provides a selective one-step synthesis for acylating the xcex5-amino group of proinsulin, insulin and insulin analogs. It is quite surprising that the invention is able to selectively acylate the xcex5-amino group in a one step process in high yield. Thus, the invention eliminates the need to protect and subsequently deprotect other amino groups of the protein. The invention provides more efficient and less expensive means of preparing xcex5-amino acylated insulin derivatives.
The present invention provides a process of selectively acylating proinsulin, insulin, or an insulin analog having a free xcex5-amino group and a free xcex1-amino group with a fatty acid, which comprises reacting the xcex5-amino group with a soluble activated fatty acid ester under basic conditions in a polar solvent.